Effect of maraviroc on non-R5 tropic HIV-1: refined analysis of subjects from the phase IIb study A4001029.

We characterized maraviroc susceptibility of dual/mixed tropic viruses from subjects enrolled onto phase IIb study A4001029. Maraviroc baseline plasma samples from 13 multidrug-experienced subjects were sequenced and the HIV-1-env gene cloned into pNL4.3Δenv to obtain recombinant viruses. The V3 region was sequenced by the Sanger method and ultradeep sequencing. By analysing subjects having a weighted optimized background therapy susceptibility (wOBT) score of <1, 3/7 subjects were characterized by good in vivo and in vitro response to maraviroc therapy. Molecular docking simulations allowed us to rationalize the maraviroc susceptibility of dual/mixed tropic viruses. A subset of subjects with dual/mixed tropic viruses responded to maraviroc. Further investigations are warranted of CCR5 antagonists in subjects carrying dual/mixed tropic virus that explore the feasible use of maraviroc in subjects that is potentially larger than those infected with a pure R5 virus.

The lowest X4 Geno2Pheno false-positive rate is associated with greater CD4 depletion in HIV-1 infected patients.

Through this study we evaluated whether the HIV-1 tropism determined by genotypic analysis correlates with HIV-1 markers, such as CD4 cell count and plasma HIV-RNA. The analysis was performed on 1221 HIV-1 B-subtype infected patients with an available V3 sequence (all maraviroc naive). Of them, 532 were antiretroviral therapy (ART) naive and 689 ART experienced. Tropism determination was performed by using the geno2pheno (co-receptor) algorithm set at a false-positive rate (FPR) of 10% and 2%. Potential associations of FPR with CD4 cell count and viraemia were evaluated. Association of V3 mutations with genotypic-determined tropism was also evaluated according to different FPR ranges. About 26% of patients (either ART naive or ART experienced) were infected by X4-tropic viruses (using the classical 10% FPR cut-off). However, a significantly lower proportion of ART-naive patients had FPR ≤ 2% in comparison with ART-experienced patients (4.9% vs. 12.6%, respectively, p <0.001). The risk of advanced HIV-1 infection (with CD4 cell count ≤ 200 cells/mm(3)) was significantly greater in X4-infected patients, either ART-naive (OR (95% CI)), 4.2 (1.8-9.2); p 0.0006) or ART-experienced (2.3 (1.4-3.6); p 0.0003), with FPR set at 2% (but not at 10%). This finding was confirmed by multivariable logistic analysis. No relationship was found between viraemia and FPR ≤2%. Some X4-related mutations were significantly associated with FPR ≤2% (ART-naive patients, S11R, Y21V, G24K and G24R, p ≤0.001; ART-experienced patients, Y7K, S11R, H13Y, p ≤0.002). In conclusion, these findings show that within the context of genotypically-assessed CXCR4 tropism, FPR ≤2% defines (far better than 10%-FPR) a viral population associated with low CD4 rank, with potentially greater cytopathic effect, and with more advanced disease.

[Controversial issues in the Giornale Italiano di Nefrologia: how to treat patients with focal segmental glomerular sclerosis]. / Le contoversie del Giornale Italiano di Nefrologia: come trattare i pazienti con glomerulosclerosi focale segmentaria?

Primary focal segmental glomerular sclerosis (FSGS) commonly presents with nephrotic syndrome. Spontaneous remission is rare and persistent nephrotic syndrome is a marker of poor prognosis. For this reason, obtaining remission using drugs with minimal side effects is desirable. The treatment of FSGS, however, represents a challenge. Not only is there a lack of prospective controlled trials, but FSGS is a syndrome of unknown pathophysiology, generally treated with drugs having a mechanism of action that is poorly understood in this setting, the use of which has often drawn criticism because it is based on empirical assumptions rather than pathogenetic evidence. At present, corticosteroids are the standard first-line approach in patients with idiopathic FSGS. Cytotoxic agents and cyclosporin A constitute a good therapeutic option for steroid-dependent patients or frequent relapsers. Mycophenolate mofetil, rituximab and plasmapheresis should be used as rescue treatment because further studies are required to determine their safety and efficacy. Clearly, real progress in FSGS treatment can only be obtained by research focused on the pathophysiology of this disease, so that a therapeutic approach can be defined that is based on reason rather than chance.

[Nephrotic proteinuria with type 2 diabetes mellitus and autoimmune thyroiditis]. / Proteinuria nefrosica in paziente con diabete mellito di tipo II e tiroidite autoimmune.

This study reports on a 67-year-old man, suffering from type 2 diabetes mellitus for 11 years along with arterial hypertension and autoimmune thyroiditis, in whom nephrotic proteinuria was detected together with a mild reduction in GFR. No autoantibodies or monoclonal proteins were detected in blood and urine. Renal biopsy material examined by light microscopy, immunofluorescence and electron microscopy showed AL amyloidosis. This case underlines the role of renal biopsy in patients with type 2 diabetes mellitus, in whom renal diseases other than diabetic nephropathy may occur frequently.

[Thin glomerular basement membrane disease]. / La nefropatia a membrane basali sottili.

Thin glomerular basement membrane disease (TBMD) is a hereditary nephropathy characterized by thinning of the glomerular basement membrane evinced by electron microscopy and, clinically, by isolated hematuria without extrarenal manifestations. Familial aggregation is found in 50-60% of cases, with autosomal dominant transmission. TBMD is considered to belong to the type IV collagen spectrum of diseases, since heterozygous mutations of the COL4A3 or COL4A4 gene have been detected in more than 30% of patients. The disease is found in 1-2% of biopsies, but the prevalence in the general population may be higher. The differential diagnosis with Alport's syndrome may be difficult and requires accurate family investigations, immunohistochemical evaluation of type IV collagen alpha chains in renal tissue and, if appropriate, genetic studies. Progression towards chronic renal failure, although rare, has been reported in some patients, and may be related to the phenotypical variability of COL4A3/COL4A4 mutations, to a missed Alport syndrome, or to superimposed glomerular disease. Patients suffering from TBMD and affected relatives should be periodically examined for signs of disease progression and informed about the possibility of transmitting the autosomal recessive form of Alport's syndrome.

Inhibition of HIV-1 replication in macrophages by red blood cell-mediated delivery of a heterodinucleotide of lamivudine and tenofovir.

Homo- and heterodimers of nucleoside/nucleotide analogues as reverse transcriptase inhibitors are effective on HIV-1-infected human monocyte-derived macrophages (M/M) compared to the single drugs or their combination. Since the combined treatment of lamivudine (3TC) and tenofovir ((R)PMPA) has an antiretroviral efficacy and a synergic effect respect to separate drugs, the heterodinucleotide 3TCpPMPA was synthesized. A single administration of the dimer as free drug or 3TCpPMPA-loaded RBC selectively targeted to M/M was able to almost completely protect macrophages from "de novo" infection.

Antiviral profile of HIV inhibitors in macrophages: implications for therapy.

Macrophages (M/M) are identified as the second cellular target of HIV and a crucial virus reservoir. M/M are persistently infected cells and not susceptible to the HIV cytophatic effects typical of infected CD4+ T-lymphocytes. HIV replication in M/M is a crucial pathogenetic event during the whole course of the disease. Moreover, the dynamics of HIV-1 replication and cumulative virus production is quite different in M/M and CD4+ T-lymphocytes in the presence or in the absence of antiviral drugs. Thus, for their unique cellular characteristics, the activity of anti-HIV compounds could be different in M/M than in CD4+ T-lymphocytes. Indeed, nucleoside analogues inhibitors of HIV-reverse transcriptase (NRTIs) show potent antiviral activity in macrophages, although the limited penetration of these compounds in sequestered body compartments and the scarce phosphorylation ability of macrophages, suggest that a phosphate group linked to NRTIs may confer a greater anti-HIV activity in such cells. The antiviral activity of non-nucleoside reverse transcriptase inhibitors (NNRTIs) in macrophages is similar to that found in CD4-lymphocytes. Interestingly, protease inhibitors (PIs), acting at post-integrational stages of virus replication, are the only drugs able to interfere with virus production and release from macrophages with established and persistent HIV infection. For these reasons, a careful analysis of the distribution of antiviral drugs, and the assessment of their activity in cells of macrophage lineage, represent key factors in the development of therapeutic strategies aimed to the treatment of the HIV-infected patients.

HIV/HCV co-infection: the magnitude of the problem.

Hepatitis C virus (HCV) and human immunodeficiency virus (HIV) share routes of transmission, therefore their coinfection is relatively common. Nevertheless, the clinical relevance of this event has been minimal until few years ago when, due to the increased survival of HIV-infected individuals (favoured by highly active antiretroviral therapy) morbility and mortality caused by pathologies not strictly related to HIV (such as HCV infection) raised sharply. Despite differences in their general characteristics (including lifecycle, target cells, and type of persistence in the infected host) a remarkable level of interaction exists between HCV and HIV; this makes the progression of both liver disease and immunological damage easier and more rapid. A therapeutic approach to HIV/HCV coinfection thus requires the utilization of drugs and strategies effective against both viruses, yet, timing, drug types, and effective combinations still remain poorly defined. New and innovative studies specifically focused on HIV/HCV coinfection are thus warranted to increase the knowledge about their interaction, and define therapeutic strategies aimed to the best management of the infection by both viruses during coinfection.

Changes in coagulation and markers of fibrinolysis in horses undergoing colic surgery.

Activation of coagulation can be frequently found in horses with colic. However, it has also been demonstrated as a sequela of surgical trauma alone in humans. The purpose of the present study was to determine changes in coagulation and fibrinolysis in horses that underwent colic surgery and to evaluate whether these changes were secondary to the colic or the surgery and wound healing. Thirty horses that underwent colic surgery with uncomplicated recovery were included. Ten horses with a Forssell's procedure served as control group with a standardized surgical trauma. Besides daily physical examinations during the observation period of 10 days, activated partial thromboplastin time (aPTT), prothrombin time and thrombin time as well as fibrin monomer (FM), D-Dimer (DD) and antithrombin (AT) III were determined. Compared with the control group the aPTT was the only standard coagulation test that was significantly prolonged before and after the event of colic surgery. After surgery, hyperfibrinogenaemia occurred in all groups. In colic groups FM and DD concentrations were within reference range at admission,and were significantly greater than in control horses after surgery. AT III activity decreased after colic surgery, but did not change in the control group. It was concluded that an activated coagulation state after colic surgery has to be expected, resulting not only from the colic disease, but also from the event of surgery.

Macrophages: a crucial reservoir for human immunodeficiency virus in the body.

The replication of Human Immunodeficiency Virus (HIV) in cells of macrophage lineage represents a key pathogenetic event of the neurological damages typically found during the course of this disease. Macrophages are persistently infected cells and thus not susceptible to the cytophatic effect typical of infected activated CD4-lymphocytes. The resistance of macrophages to HIV infection is at least in part mediated by the autocrine production of the nerve growth factor (NGF), a neurokine able to sustain the survival of some cells of bone marrow origin, including monocyte-derived macrophages. This anti-apoptotic effect of NGF in HIV-infected macrophages can be even more relevant at the central nervous system level, where many cells are able to physiologically produce NGF, thus further increasing the survival of macrophages infected by HIV, and enhancing the damages that these cells may induce upon bystander neurons. The proapoptotic effect of soluble factors released by HIV-infected macrophages may heavily affect the survival and functions also of astrocytes, that in turn become unable to sustain neuronal homeostasis. Taken together, this information supports the importance of therapeutic attempts aimed at attacking virus replication in infected macrophages and/or to selectively eliminate these chronically infected and persistently virus-producing cells.

The potency of acyclovir can be markedly different in different cell types.

Acyclovir is an acyclic guanine analog with a considerable activity against herpes simplex viruses. We studied the antiherpetic activity of acyclovir in macrophages and fibroblast cell lines. Utilising a plaque reduction assay we found that acyclovir potently inhibited the HSV-1 replication in macrophages (EC50) = 0.0025 microM) compared to Vero (EC50 = 8.5 microM) and MRC-5 (EC50 = 3.3 microM) cells. The cytotoxicity of acyclovir was not detected at concentrations < or = 20 microM, thus the selective index in macrophages was >8000. This marked difference in antiherpetic activity between macrophages and fibroblasts was not observed with Foscarnet and PMEA. We suggest that this potent antiviral effect of acyclovir is mainly due to a proficient phosphorylation of the drug and/or a favourable dGTP/acyclovir triphosphate ratio in macrophage cells.

Erythrocyte-mediated delivery of a new homodinucleotide active against human immunodeficiency virus and herpes simplex virus.

Monocyte-derived macrophages (MDMs) play a central role in the pathogenesis of infection by human immunodeficiency virus (HIV-1) and represent one of the main reservoirs of the virus in the body. In addition, MDMs can easily be infected by various herpes viruses, including herpes simplex virus type 1 (HSV-1). We have synthesized a new antiviral agent (Bis-PMEA) that consists of two 9-(2-phosphonylmethoxyethyl)adenine (PMEA) molecules bound by a phosphate bridge. This nucleotide analogue, like the parent compound PMEA, has strong and selective activity against HIV-1 and HSV-1. A drug-targeting system previously developed in our laboratory was used for the selective delivery of these drugs to macrophages. Bis-PMEA and PMEA were encapsulated into autologous erythrocytes by a procedure of hypotonic dialysis and isotonic resealing. Loaded erythrocytes were modified to increase their recognition and phagocytosis by human macrophages. By administering Bis-PMEA-loaded erythrocytes to macrophages, 47% of Bis-PMEA and 28% of PMEA was still present 10 days after phagocytosis; in contrast, only 12% of PMEA was found in macrophages receiving PMEA-loaded erythrocytes. Bis-PMEA-loaded erythrocytes were then added to macrophages infected with HIV-1 and HSV-1 and their antiviral activity evaluated. Remarkable protection was obtained against HIV-1 and HSV-1 infection (95 and 85%, respectively). Therefore, Bis-PMEA acts as an efficient antiviral prodrug that, following selective targeting to macrophages by means of loaded erythrocytes, can protect a refractory cell compartment.

Inhibition of HIV-1 replication in macrophages by red blood cell-mediated delivery of a heterodinucleotide of azidothymidine and 9-(R)-2-(phosphono methoxypropyl)adenine.

Monocyte-derived macrophages (M/M) are considered important in vivo reservoirs for different kinds of viruses, including HIV. Hence, therapeutic strategies are urgently needed to protect these cells from virus infection or to control viral replication. In this paper, we report the synthesis, target delivery and in vitro efficacy of a new heterodinucleotide (AZTpPMPA), able to inhibit HIV-1 production in human macrophages. AZTpPMPA consists of two established anti-HIV drugs [zidovudine (AZT) and tenofovir (PMPA)] chemically coupled together by a phosphate bridge. This drug is not able to prevent p24 production when administered for 18 h to M/M experimentally infected with HIV-1 Bal (inhibition 27%), but can almost completely suppress virus production when given encapsulated into autologous erythrocytes (inhibition of p24 production 97%). AZTpPMPA is slowly converted to PMPA, AZT monophosphate and AZT (36 h half-life at 37 degrees C) by cell-resident enzymes. Thus AZTpPMPA should be considered a new prodrug of AZT and PMPA that is able to provide stechiometric amounts of both nucleoside analogues to macrophage cells and to overcome the low phosphorylating activity of M/M for AZT and the modest permeability of PMPA.

Primary macrophages infected by human immunodeficiency virus trigger CD95-mediated apoptosis of uninfected astrocytes.

Infection of macrophages (M/M) by human immunodeficiency virus (HIV) is a main pathogenetic event leading to neuronal dysfunction and death in patients with AIDS dementia complex. Alteration of viability of neurons and astrocytes occurs in vivo even without their infection, thus it is conceivable that HIV-infected M/M may affect viability of such cells even without direct infection. To assess this hypothesis, we studied the effects of HIV-infected M/M on an astrocytic cell-line lacking CD4-receptor expression. Exposure to supernatants of HIV-infected M/M triggers complete disruption and apoptotic death of astrocytic cells. This effect is not related to HIV transmission from infected M/M, because HIV-DNA and p24 production in astrocytic cells remained negative. Apoptotic death of astrocytes is mainly mediated by Fas ligand released in supernatants of HIV-infected M/M (as demonstrated by complete reversal of such phenomenon by adding neutralizing antibodies against CD95 receptor). Treatment of astrocytic cells with recombinant (biologically active) Tat induces < 10% apoptosis, and gp120 was totally ineffective. Treatment of HIV-infected M/M with AZT completely reverses the proapoptotic effect of their supernatants on astrocytes, thus demonstrating that productive virus replication within M/M is required for the induction of astrocytic cell death. Taken together, data suggest that homeostasis of astrocytes may be affected by HIV-infected M/M in the absence of productive infection of target cells. This phenomenon may help to explain the cellular damage found in HIV-infected patients also in areas of the brain not strictly adjacent to HIV-infected M/M.

A new acyclic heterodinucleotide active against human immunodeficiency virus and herpes simplex virus.

The most common therapies against human herpes virus (HSV-1) and human immunodeficiency virus (HIV-1) infectivity are based on the administration of nucleoside analogues. Acyclovir (ACV) is the drug of choice against HSV-1 infection, while the acyclic nucleoside phosphonate analogue PMPA has shown marked anti-HIV activity in a phase I and II clinical studies. As monocyte-derived macrophages are assumed to be important as reservoirs of both HSV-1 and HIV-1 infection, new approaches able to inhibit replication of both viruses in macrophages should be welcome. ACVpPMPA, a new heterodinucleotide consisting of both an antiherpetic and an antiretroviral drug bound by a phosphate bridge, was synthesized and encapsulated into autologous erythrocytes modified to increase their phagocytosis by human macrophages. ACVpPMPA-loaded erythrocytes provided an effective in vitro protection against both HSV-1 and HIV-1 replication in human macrophages.

[Clinical symptoms of and diagnostic possibilities for hypophyseal adenoma in horses]. / Klinische Symptomatik und diagnostische Möglichkeiten des Hypophysenadenoms beim Pferd.

Hirsutism was the most often observed symptom in horses with a pituitary gland tumor and was present in all 13 examined horses. Other symptoms were atrophy of muscles (n = 10), hyperhidrosis (n = 8), polyuria/polydipsia (n = 5), bulging or supraorbital fat (n = 3), polyphagia (n = 2), apathy (n = 2) and seizures (n = 2). Laminitis was the most frequently observed concurrent disease (n = 8). Hyperglycaemia (mean, 9.9 +/- 3.71 mmol/l) in 13 horses and glucosuria (median, 55 [range, 2-55] mmol/l) in 7 horses were the most important laboratory results. The dexamethasone suppression test was positive in all tested horses (n = 9) 20 h after administration of dexamethasone. The pituitary gland tumor was visible in every case underwent computed tomography (n = 7). From these results it can be concluded that a pituitary gland tumor can be suspected based on typical clinical signs. Hyperglycaemia and glucosuria support the preliminary diagnosis and a positive dexamethasone suppression test allows a final diagnosis.

Synthesis and biological application of a new heterodinucleotide with both anti-HSV and anti-HIV activity.

A new antiviral drug with both anti-HSV and anti-HIV activity was synthesized by coupling Acyclovir and the acyclic nucleoside phosphonate (R)PMPA. The heterodinucleotide ACVpPMPA encapsulated into autologous erythrocytes was added to human macrophages providing an effective in vitro protection from HSV-1 and HIV-1 replication.

Clinical implications of HIV dynamics and drug resistance in macrophages.

Macrophages are widely recognized as the second major target of HIV in the body. The cellular characteristics of such resting cells markedly affect the dynamics of virus lifecycle, that is slower but far more prolonged that in lymphocytes. In addition, the limited concentrations of endogenous nucleotide pools in macrophages downregulate the enzymatic activity of reverse transcriptase. As a consequence, both the anti-HIV activity and the development of resistance to antiviral drugs in macrophages are substantially different than those found in activated lymphocytes. These peculiar characteristics of virus replication and efficacy of antiviral drugs in macrophages have a natural in vivo counterpart in extralymphoid tissues, where macrophages account for the majority of cells infected by HIV. Furthermore, the replication of HIV in macrophages of testis and central nervous system is far less affected by antiviral drugs than in lymph nodes, because of the presence of natural barriers that markedly diminish the concentration of such drugs. For all these reasons, HIV infection of macrophages should be taken into account in therapeutic strategies aimed to achieve an optimal therapeutic effect in all tissue compartments where the virus hides and replicates.

[Correlation between HIV-inhibiting drug activity in human macrophages and clinical outcome]. / Attività di farmaci inibitori del virus dell'immunodeficienza umana in macrofagi umani e correlazione con l'efficacia clinica.

PURPOSE: To assess the comparative efficacy of drugs inhibitors of human immunodeficiency virus (HIV) in human macrophages and lymphocytes, and to correlate the results with the clinical outcome. MATERIALS AND METHODS: Human primary macrophages and lymphocytes were infected with HIV in the presence of the following HIV inhibitors, all currently in clinical use: zidovudine, stavudine, zalcitabine, didanosine, lamivudine, PMEA, PMPA (all inhibitors of HIV reverse transcriptase), saquinavir and U-75875 (inhibitors of HIV protease). RESULTS: All reverse transcriptase inhibitors tested showed a markedly higher antiviral activity in macrophages than in lymphocytes. Also protease inhibitors have a substantial anti-HIV activity in macrophages, yet their efficacy is markedly diminished if the drugs are added to macrophage culture after HIV, that is when the virus has established a chronical infection. Under these experimental conditions, however, only protease inhibitors among all HIV-inhibitors in clinical use are able to decrease virus replication in chronically-infected macrophages. CONCLUSIONS: The results have strong clinical implications, due to the important role of macrophages in the pathogenesis of HIV infection. Macrophages are the major source of HIV at extralymphoid tissue levels, particularly in the central nervous system, where the blood-brain barrier strongly limits the penetration of antiviral drugs. For these reasons, only drugs, like stavudine and zidovudine, provided with good anti-HIV activity in macrophages, and reasonable barrier penetration have substantial chances to be effective in the central nervous system, and thus affect virus replication in a sanctuary where HIV hides and replicates out of the control of the immune system.

Macrophage protection against human immunodeficiency virus or herpes simplex virus by red blood cell-mediated delivery of a heterodinucleotide of azidothymidine and acyclovir.

Human herpesvirus (HSVs) are distributed worldwide and are among the most frequent causes of viral infection in HIV-1-immunocompromised patients. Hence, therapeutic strategies able to inhibit HSV-1 and HIV-1 replication are sorely needed. Until now, the most common therapies against HSV-1 and HIV-1 infectivity have been based on the administration of nucleoside analogs; however, to be active, these antiviral drugs must be converted to their triphosphorylated derivatives by viral and/or cellular kinases. At the cellular level, the main problems involved in the use of such drugs are their limited phosphorylation in some cells (e.g., antiretroviral drugs in macrophages) and the cytotoxic side effects of nucleoside analog triphosphates. To overcome these limitations, a new heterodinucleotide (AZTp2ACV) consisting of both an antiretroviral and an antiherpetic drug, bound by a pyrophosphate bridge, was designed and synthesized. The impermeant AZTp2ACV was encapsulated into autologous erythrocytes modified to increase their recognition and phagocytosis by human macrophages. Once inside macrophages, metabolic activation of the drug occurred. The addition of AZTp2ACV-loaded erythrocytes to human macrophages provided effective and almost complete in vitro protection from HIV-1 and HSV-1 replications, respectively. Therefore, AZTp2ACV acts as an efficient antiviral prodrug following selective targeting to macrophages by means of loaded erythrocytes.